Worldwide, nearly 300,000 diseased heart valves are replaced annually, most of them with devices that include mechanical valves, devices made from non-living biological tissues or viable human allografts. Durability of heart valve replacements is limited to 15-20 years mostly due to coagulation risks, endocarditis, degeneration, calcification and failure to grow and remodel. This study is highly relevant to public health because heart valve disease is a very important chapter of cardiovascular diseases in adults and children. Our long-term objective is to develop living tissue-engineered valves that will last a life-time, will not be prone to complications, will have the ability to grow and remodel and thus ultimately impacting thousands of patients. Our innovative proposal acknowledges the vital importance of four issues that are unique to our approach: i) Constructs made from partially stabilized collagenous scaffolds, ii) Anatomically analogous 3-D heart valve shapes made form tri-layered structures that mimic the native heart valve histo-architecture, iii) Autologous multipotent mesenchymal stem cells for repopulation and remodeling and iv) Mechanical and biochemical cues to induce stem cell differentiation into valvular cells capable of maintaining matrix homeostasis. To accomplish these goals, we propose to develop partially stabilized collagen scaffolds that structurally and functionally mimic the aortic valve fibrosa, ventricularis and spongiosa layers, to assemble them into tri-layered constructs shaped in the form of natural heart valves and populate them with human mesenchymal stem cells. Constructs will be mounted in a bioreactor to induce differentiation of stem cells into analogues of valvular interstitial cells and promote remodeling. In Specific Aim 1, collagen layers to be used as fibrosa and ventricularis layers will be prepared from decellularized pericardium and lightly cross-linked to allow for controlled biodegradation. For the spongiosa layer, highly porous collagen scaffolds will be prepared from decellularized, elastase- treated arteries and enriched with valve-specific glycosaminoglycans. Scaffolds will be then assembled into histologically analogous tri-layered structures (fibrosa / spongiosa / ventricularis) and shaped into constructs resembling native aortic roots by molding on silicone rubber casts. Engineered aortic roots will be characterized by advanced mechanical analysis and their function evaluated in a pulsatile valve duplicator. In Specific Aim 2, we will prepare human mesenchymal stem cells. Stem cells will be then seeded onto collagen gels and subjected to controlled load regimes in a FlexerCell system. We will evaluate phenotypic changes and ability of stimulated stem cells to differentiate into valvular interstitial cells. In Specific Aim 3, we will encase spongiosa layer within the tri-layered scaffold, seed the scaffolds with stem cells, and subject constructs to in vitro cycling within a bioreactor. We will evaluate cell differentiation and matrix remodeling at various time-points in dynamic conditions.